Project Summary It has been estimated that ~3% of the word population is infected with hepatitis C virus (HCV). Recent studies have identified a number of neutralizing epitopes on the HCV E1 and E2 glycoproteins and demonstrated with atomic structures how broadly neutralizing antibodies (bnAbs) interact with these epitopes. The 2.65 crystal structure of HCV E2 core (E2c) in complex with a bnAb (AR3C) revealed atomic details of this long-sought target, providing a structural basis for rational design of HCV vaccines. In Project 2 of this U19 proposal, we will combine the latest findings in HCV structural biology and cutting-edge technologies in computational protein design and next-generation sequencing (NGS) of B-cell repertoire to facilitate HCV immunogen design for the induction of bnAbs in vaccination. The specific aims are: (1) to develop epitope-focused immunogens and E2 core-based immunogens. Crystal structures of E2 and E1 neutralizing epitopes in complex with bnAbs have been determined recently. We hypothesize that heterologous protein scaffolds presenting a grafted HCV epitope can induce cross-neutralizing antibodies to the epitope. We also hypothesize that the E2c domain can be optimized as a subunit vaccine immunogen to induce cross-neutralizing antibodies to the conserved epitopes presented on the E2 surface. Based on the crystal structure of E2c, we will shorten the variable loops, engineer the surface N-linked glycans, and introduce space-filling mutations to the front layer region to stabilize the E2c conformation. Successfully designed and expressed HCV antigens will be displayed multivalently on ferritin particle and bacteriophage Q? VLP. (2) to use NGS repertoire profiling to assess antibody response in mouse and non-human primate immunization. Promising immunogen candidates will be first tested in mice. In collaboration with the Law lab, an array of HCV-specific immunological assays will be available and performed to map the serum antibody responses. A small set of designed immunogens that can induce cross-neutralizing antibodies in mice will be tested in non-human primates (NHPs) at the Southwest National Primate Research Center (SNPRC). Flow cytometry-based single B-cell sorting and microfluidics-based B-cell encapsulation will be utilized to isolate HCV-neutralizing antibodies (nAbs). NGS-based repertoire profiling will be used to obtain a quantitative readout of antibody response during immunization and to trace the lineage development of HCV nAbs. The antibody repertoire profiles will be used to guide immunogen optimization and to compare different immunogen candidates. The research proposed in Project 2 will therefore be complementary to and will benefit from the human sample analysis, epitope mapping, and testing of the Novartis E1E2 vaccine in Project 1, and together constitute the center for studying hepatitis C virus antibody responses and vaccine antigens.